1. Field of the Invention
The present invention relates to the fields of glycan biomarkers and serum anti-glycan antibody signatures of prostate disease.
2. Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. The discussion below should not be construed as an admission as to the relevance of the information to the claimed invention or the prior art effect of the material described.
Recent studies suggest that the clinical outcome of prostate cancer highly correlates with the presence of aggressive cancer, including the high volume or percentage of Gleason grade 4/5 cancer and the rapid progressive small cancer in a subject. The Gleason grade is a system to categorize the severity of the cancer based exclusively on differentiation, defined as the resemblance of the cancer cells to the architectural structure of normal prostate glands. The system scores cancers from the well-differentiated grade 1 to the poorly differentiated grade 5. While cells of grade 1 carcinoma closely resemble normal prostate, those of higher grades progressively demonstrate loose aggregation, infiltration of glands into neighboring stroma, and nuclear morphological changes. Particularly, grade 4/5 carcinoma presents a distinct loss of recognizable glandular pattern, replaced by mere sheets of cells with irregular cyto-structures. To determine the final Gleason score, the pathologist examines the biopsy specimen, attempts to grade the two most prominent patterns, and sums up the individual scores.
Given the fact that the presence of aggressive prostate cancer is correlated with the morbidity and mortality of the disease, it is crucial to establish assays not only to detect the presence of prostate cancer but also to differentially detect the presence of aggressive prostate cancer. By reviewing a large amount of experimental and clinical data, Stamey and his colleagues concluded that the percentage of grade 4/5 cancer in the primary tumor is the most significant prognostic marker. However, neither serum PSA levels nor biopsies correctly reveal the volume or grade of cancer in the prostate. Measurement of the percentage of grade 4/5 cancer in a subject can only be achieved after radical prostatectomy. The grade denominators in comparative studies have major influences on the resultant list of genes found to be differentially expressed in prostate cancer. Therefore, it is critical in comparative genetic profiling studies that the histopathology of the tissues be rigorously and precisely defined.
Secondly, investigators rarely distinguish between the expression of a proposed cancer marker in TZ (transition zone) cancer versus PZ (peripheral zone) cancer. The clinical behavior of TZ cancer is very different from that of PZ cancer, and the ideal marker will allow differential diagnosis of these cancers of different zones. Furthermore, the Gleason grades of the cancers are often not precisely defined, but are given instead as a score. A score of 7 is imprecise and does not distinguish a cancer containing 5% grade 4 and 95% grade 3 from one with 5% grade 3 and 95% grade 4. As Stamey and colleagues have shown, the risk of recurrence is vastly different between these two types of cancer, both with a “score” of 712. The biochemical PSA cure rate after radical prostatectomy for men with tumors containing 5% grade 4/5 cancer approaches 90%, whereas that for men with 95% grade 4/5 cancer is about 10%. We presume that genetic changes are the basis of the increased aggressiveness associated with increasing percentages of grade 4/5 cancer, and that grade is a critical variable in genetic profiling studies. Because of the problems described above, many of the published prostate “cancer” markers identified from genetic profiling studies would fail to remain as markers if subjected to more rigorous investigation. In any case, information from genetic profiling has yet to result in a serum marker for prostate cancer.
In 2005, it was reported that autoantibodies against peptides derived from prostate cancer tissue might be used as the basis of a screening test for prostate cancer20. Investigators from the University of Michigan used a phage-display library derived from prostate cancer tissue to develop a phage protein microarray to analyze serum samples from men with prostate cancer and controls for autoantibodies. From the initial results, a 22-phage-peptide detector was developed that had 88% specificity and 82% sensitivity in discriminating between the group with prostate cancer and the control group. This panel performed better than PSA in discriminating the two groups. The “autoantibody signature” did not discriminate among different clinical or pathological features such as Gleason grade. It is interesting to note that four of the phage clones represented known proteins, whereas the others were generated from untranslated sequences.
The carbohydrate chains, N-glycans, attached to the single glycosylation site on PSA from normal sera or seminal fluid, as compared with those on PSA from a prostate cancer cell line or the cancer tissues of patients, have been the subject of studies by the groups of Robbins, deLlorens and Fukuda21-23. The N-glycans of normal PSA are predominantly biantennary whereas those described on PSA from prostate cancer cells are predominantly tri- and tetra-antennary. The malignant glycoform of PSA from prostate cancer tissue was found to be preferentially bound by the sialyl α2-3-galactose-specific plant lectin Maackia amurensis23. To overcome the ambiguities associated with the overlap of PSA levels in men with BPH versus cancer, as determined using antibodies raised to the whole glycoprotein, Danishefsky and colleagues24 have undertaken chemical synthesis of di- tri- and tetra-antennary N-glycans with a view to attaching these to the PSA protein, and thus being able to raise specific antibodies to the normal and malignant glycoforms of the glycoprotein.
Carbohydrate chains are prominently displayed at the surface of cells, attached to glycoproteins and glycolipids. All are potential antigens. Work with naturally occurring and hybridoma antibodies has served to single out carbohydrate antigens that distinguish normal from cancer cells 26-29. Such tumor-associated antigens have been those expressed exclusively on glycoproteins such as the core regions of O-glycans (T, Tn and sialyl-Tn antigens) or exclusively glycolipids (gangliosides) or shared between glycoproteins and glycolipids: the branched and linear type II backbone regions (I and i antigens), and peripheral regions (blood group A, B, H Lewisa (Lea) and Leb antigens; also blood group-related antigens Lex, Ley, sialyl-Lex and sialyl-Lea, in cells that do not normally express them).
Glycoprotein sugar chain determinants that have been shown to be associated with prostate cancer include those that are formed by very short O-glycans, the T antigen30, 31 also sialyl Tn, and Tn and also globo-H32. These have been among targets selected for immunotherapy in clinical trials24, 33-35 of prostate cancer. Other manifestations of prostate cancer have been incomplete oligosaccharide synthesis leading to the loss of predicted blood group antigens36 as on glycolipids37.
Reverse changes, i.e., increased carbohydrate antigenicities, have also been observed in documented prostatic carcinomas38, with preservation or increased expression of type II backbone sequences, and with further glycosylation and expression of the difucosylated Ley or the monofucosyl, monosialyl compound sialyl-Lex, Globo H, blood group H, and Leb39 and also of type I backbone based sialyl sialyl-Lea40. In another study, up-regulation of the expression of oligosaccharide sialyl-Lex was considered a new prognostic parameter in metastatic prostate cancer41.
In summary, a current challenge in prostate cancer research is to identify novel biomarkers, especially serum biomarkers that allow detection of the presence of prostate cancer and determine its aggressiveness. Although considerable efforts have been made, there is so far no solid progress in identification of protein-based novel serum biomarkers of prostate cancer. Key aspects of the present carbohydrate based biomarkers, as discussed below, are a) these carbohydrate structures are immunogenic and thereby elicit specific autoantibodies in prostate cancer subjects and b) detection of autoantibodies targeting these carbohydrate structures allows detection of prostate cancers, especially the presence of aggressive Gleason 4/5 cancers, in a subject. A high-throughput platforms of carbohydrate microarrays as described here will greatly facilitate these tests. In addition, the present invention concerns certain lectins which have been found to bind selectively to prostate markers in different conditions.